Tnfα clone mp6 xt22

Manufactured by BD

Sourced in Japan

TNFα (clone MP6-XT22) is a mouse monoclonal antibody that specifically recognizes the tumor necrosis factor alpha (TNFα) protein. It can be used for the detection and quantification of TNFα in various research applications.

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TNFα (MP6-XT22; (10 citations) MP6-XT22 (9 citations) Clone MP6-XT22) (3 citations) Anti-TNFα (clone MP6-XT22). (3 citations) Anti-mouse TNFα (clone MP6-XT22), (3 citations) TNFα-PE (clone MP6-XT22) (2 citations)

6 protocols using tnfα clone mp6 xt22

Isolation and Characterization of Liver-Infiltrating Immune Cells

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The isolation of liver-infiltrating mononuclear cells was conducted as previously described8 (link). Mononuclear cells were stained with fluorochrome-conjugated antibodies, including CD4 (clone RM4-5), CD8α (clone 53-6.7), TCRβ (clone H57-597), F4/80 (clone T45-2342), CD11c (clone HL3), CD19 (clone 1D3), I-A/I-E (clone AF6-120.1), CD86 (clone GL1), IL-12 (clone C15.6), and TNFα (clone MP6-XT22) (BD Biosciences). Isotype Abs with matching conjugates were used as negative controls. For intracellular staining, cells were activated with PMA/ionomycin and processed as previously described. Live/dead cell discrimination was achieved by staining with Propidium Iodide, 10,000 events of viable cells were analyzed with the FACSCalibur Flow Cytometer (BD Biosciences), and analysis was conducted with FlowJo (Tree Star).

Arsenijevic A., Milovanovic M., Milovanovic J., Stojanovic B., Zdravkovic N., Leung P.S., Liu F.T., Gershwin M.E, & Lukic M.L. (2016). Deletion of Galectin-3 Enhances Xenobiotic Induced Murine Primary Biliary Cholangitis by Facilitating Apoptosis of BECs and Release of Autoantigens. Scientific Reports, 6, 23348.

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Cytokine Production in CD8+ T Cells

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CD8⁺ T cells isolated from the spleen of WT and Pld2^−/− mice were seeded on 12-well plates at 2 × 10⁵ cells/well and stimulated with anti-CD3/CD28 antibodies for 24 hr as described above. Brefeldin A (eBioscience) was added to the culture for the final 6 hr incubation to block cytokine secretion. Cells were collected and stained for CD8 with anti-CD8 antibody for 30 min at 4 °C, followed by fixation and permeabilization with the Intracellular Fixation and Permeabilization buffer set (eBioscience). After washing, antibodies against IL-2 (clone JES6–5H4; BD Biosciences), IFN-γ (clone XMG1.2; BD Biosciences) and TNF-α (clone MP6-XT22; BD Biosciences) were added to the cells and subjected to flow cytometry analysis, and cell number producing these cytokines was counted.

Ngo Thai Bich V., Hongu T., Miura Y., Katagiri N., Ohbayashi N., Yamashita-Kanemaru Y., Shibuya A., Funakoshi Y, & Kanaho Y. (2018). Physiological function of phospholipase D2 in anti-tumor immunity: regulation of CD8+ T lymphocyte proliferation. Scientific Reports, 8, 6283.

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Multiparametric Immune Cell Profiling

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Lymphocytes were counted and subjected to viability staining (Fixable Aqua Dead Cell staining, Life Technologies, #L34957) and subsequently to receptor Fc blockade (BD #553142). Staining was performed using the following antibodies: CD4 (clone RM4-4, BD), CD45 (clone 30-F11 eBioscience), CD45.1 (clone A20, BD), CD45.2 (clone 104, BD) CD8a (clone 53-6.7, eBioscience or BD), CD8b (clone YTS156.7.7, BioLegend), CD11b (clone RM2817, Thermo Fisher), Ly6c (clone AL-21, BD), CD3ε (clone 145-2C11, BD), CD19 (clone 1D3, BD), CD90.2 (53-2.1, BD), CD127 (clone A7R34, BD), CX3CR1 (clone SA011F11, BioLegend), CXCR3 (clone CXCR3-173, Biolegend), CD103 (clone 2E7, eBioscience or Biolegend), CD69 (clone H1.2F3, BD), TCR-β (clone H57-597, BD, TCR-γδ (clone eBio-GL3, eBioscience). H2-K^b:SIINFEKL or H2-M3:fMIGWII tetramers were either produced in house or, for the former, obtained thorugh the NIH tetramer core. Samples were fixed (IC Fixation Buffer, eBioscience), washed, resuspended in FACS buffer and acquired with a LSRII flow cytometer (BD) either immediately or on the following day. For intracellular staining, the following antibodies were used: IFN-ϒ (clone XMG1.2, BD), TNF-α (clone MP6-XT22, BD) in Permeabilization buffer (eBioscience).

Becattini S., Littmann E.R., Seok R., Amoretti L., Fontana E., Wright R., Gjonbalaj M., Leiner I.M., Plitas G., Hohl T.M, & Pamer E.G. (2020). Enhancing mucosal immunity by transient microbiota depletion. Nature Communications, 11, 4475.

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Comprehensive Western Blot and Microscopy Antibody Panel

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The following Western blot antibodies were purchased from Cell Signaling: rabbit anti-HA (clone C29F4), mouse anti-FLAG (clone 9A3), mouse anti-GFP (clone 4B10), rabbit anti-phospo-Erk (clone D13.14.4E), mouse anti-Total Erk (clone L34F12), and mouse anti-IκBα (clone 44D4). Secondary antibodies (mouse and rabbit IgG) were purchased from Licor.
Antibodies used for flow cytometry from BD include TNFα (clone MP6-XT22) conjugated to Alexa Fluor 700, IL-6 (clone MP5-20F3) conjugated to PE, and CD11c (clone HL3) conjugated to PeCy7. HA (clone 6E2) conjugated to Alexa Fluor 647 was purchased from Cell Signaling.
Antibodies used for microscopy include anti-HA antibody (product no. 11 867 423 001, Roche), mouse anti-LAMP1 antibody-lysosomal marker (product no. sc-18821, Santa Cruz Biotechnoogy), mouse anti-GM130 antibody-Cis-golgi marker (product no. 610822, BD Transduction laboratories), Rabbit anti-calreticulin antibody-ER marker (product no. ab 2907, abcam), mouse anti-SQSTM1 / p62 antibody- autophagy marker (product no. ab 56416, abcam) and Rabbit anti-LC3b antibody-autophagy marker (product no. 3868, Cell signaling Technology). The respective secondary antibodies used were goat anti-rat-alexa fluor 568 (product no. A-11077), and Goat anti-Rb-alexa flour 488 (product no. A1126) and Goat anti-mouse-alexa flour 488 (product no. A28175) obtained from Invitrogen/Thermofisher Scientific.

Biswas C., Rao S., Slade K., Hyman D., Dersh D., Mantegazza A.R., Zoltick P.W., Marks M.S., Argon Y, & Behrens E.M. (2018). Tyrosine 870 of TLR9 is critical for receptor maturation rather than phosphorylation-dependent ligand-induced signaling. PLoS ONE, 13(7), e0200913.

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Serum Antibody and Cytokine Measurement

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Serum levels of anti-SSA/Ro and anti-SSB/La antibodies (Alpha Diagnostics International, San Antonio, TX, USA) were determined using ELISA kits according to the manufacturer’s protocol. Serum inflammatory cytokines in mice sera were measured by sandwich-ELISA using capture antibodies against IL-1β (clone B122; eBioscience, Affymetrix Japan, Tokyo, Japan), IL-5 (clone TRFK5; eBioscience), IL-10 (clone JESS-16E3; eBioscience), IL-17A (clone 17CK15A5; eBioscience), IFN-γ (clone XMG1.2; eBioscience), TNF-α (clone TN3-19.12; BD Pharmingen), and biotinylated-detection antibodies against IL-1β (clone 13-7112-85; eBioscience), IL-5 (clone TRFK4; eBioscience), IL-10 (clone JESS-2A5; eBioscience), IL-17A (clone 17B7; eBoiscience), IFN-γ (clone R4-6A2; eBioscience), TNF-α (clone MP6-XT22; BD Pharmingen). All antibodies were diluted 1:1000 in PBS containing 10% fetal calf serum (Gibco, Thermo Fisher Scientific, Tokyo, Japan). Biotinated antibodies were detected by HRP-conjugated avidin (Vector laboratories, Burlingame, CA, USA) and visualized by 3,3′,5,5′-tetramethylbenzidine (Dako), where the reaction was stopped with 2 M H₂O₂. Microtitier plate reader (Vmax, Molecular Devices, Tokyo, Japan) at 450 nm was used to measure the optical densities.

Yanagisawa N., Ueshiba H., Abe Y., Kato H., Higuchi T, & Yagi J. (2018). Outer Membrane Protein of Gut Commensal Microorganism Induces Autoantibody Production and Extra-Intestinal Gland Inflammation in Mice. International Journal of Molecular Sciences, 19(10), 3241.

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Mediastinal Lymph Node Characterization

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Mediastinal lymph node cells were prepared and stained for surface and intracellular markers per our previous protocols (5 (link)), using antibodies specific for CD4 (clone L3T4), CD8 (clone 53-6.7), IFNγ (clone XMG1.2) and TNFα (clone MP6-XT22) (BD Biosciences).

Reeme A.E, & Robinson R.T. (2016). Dietary Vitamin D3 suppresses pulmonary immunopathology associated with late stage tuberculosis in C3HeB/FeJ mice. Journal of immunology (Baltimore, Md. : 1950), 196(3), 1293-1304.

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